Hematopoiesis, the proliferation and differentiation of blood cells from pluripotent stem cells, has been found to be regulated by a variety of cell factors (i.e. cytokines), examples of which are the interleukins (IL's) and colony-stimulating factors (CSF's).
Human interleukin-6 (IL-6), in particular, is produced by the lymphoid and other cells and plays a role in stimulating proliferation of multiple lineages of hematopoietic cells. Examples of hematopoietic activities ascribed to IL-6 include antiviral activity, stimulation of B-cells and Ig secretion, induction of IL-2 and IL-2 receptor expression, enhancement of IL-3 induced colony formation, proliferation and differentiation of T-cells, maturation of megakaryocytes, and other functions.
The pleiotropic or multifunctional nature of human IL-6 is reflected in the plurality of names used in the art [e.g., interferon-.beta..sub.2 (IFN.beta..sub.2), 26 kDa protein (26K), B-cell stimulatory factor 2 (BSF-2), hybridoma/plasmacytoma growth factor (HPGF), hepatocyte stimulating factor (HSF), cytotoxic T-cell differentiation factor (CDF)] to refer to what has been confirmed by molecular cloning to be a single protein of 212 amino acids and a molecular mass ranging from 21 to 28 kd, depending on the cellular source and preparation (see Van Snick, Ann. Rev. Immunol. 1990, 253. Recombinant human IL-6 protein has been molecularly cloned and purified to homogeneity.
The terms "IL-6" and "IL-6 protein" as used herein shall be understood to refer to a natural or recombinantly prepared protein, which may be glycosylated or unglycosylated and which has the amino acid sequence of natural human IL-6 as disclosed, for example, in published PCT application Serial No. WO 88/00206, which is incorporated herein by reference.
A well-documented inter-species activity of human IL-6 comprises stimulation of thrombocytopoiesis, i.e. the process by which megakaryocyte progenitor cells mature into megakaryocytes, from which the platelets are ultimately released into peripheral circulation (see McDonald, "The Regulation of Megakaryocyte and Platelet Production," in Concise Reviews in Clinical and Experimental Hematology, ed. by M. Murphy, AlphaMed Press, Dayton, Ohio (1992) at 167).
For example, administration of recombinant human IL-6 (hereinafter also rhIL-6) to normal mice and monkeys has been found to result in increased megakaryocyte size and elevated peripheral blood platelet counts (see, e.g., Stahl et al., Blood, Vol. 78, No. 6 Sep. 15, 1991: pp 1467-1475; Mayer et al. Exp. Hematol. 19:688-696).
IL-6 induced platelet production has also been documented in a non-human primate model of radiation-induced marrow aplasia (see, e.g., MacVittie et al., Blood, November 15,) 1992, Vol. 80, No. 10), as well as in humans subjected to ICE chemotherapy, Chang et al., Blood, id.
The platelets contribute a vital homeostatic function by adhering and coagulating on damaged tissue and by secreting factors which initiate coagulation reactions. A deficiency of platelets (thrombocytopenia) whether caused by failure of platelet production (e.g., as a result of aplastic anemia), and/or megakaryocyte depression brought on by iatrogenic drugs, chemicals or viral infections, AIDS related problems and/or platelet destruction (e.g., as a result of secondary thrombocytopenia), can be a life-threatening condition, for which the only conventional treatments have been repeated platelet transfusions, or bone marrow transplantation, both involving risks of infection and rejection.
Administration of IL-6 to a patient suffering from platelet deficiency may therefore be practiced as an endogenous means of accelerating recovery from thrombocytopenia, and even spare the need for transfusion or transplantation. IL-6 may also be used and particularly important in treating subjects in whom thrombocytopenia has been induced by irradiation or administration of drugs which interfere with hematopoiesis (see Patchen et al., Blood, Vol. 77, No. 3 (February 1), 1991: pp. 472-480).
However, administration of IL-6 therapy to a mammalian patient for purposes of obtaining the various benefits and advantages therefrom, including, in particular, stimulation of thrombocytopoiesis, or for other therapeutic purposes, is often accompanied by associated systemic changes which, at higher dosages of IL-6 or over prolonged periods of time, may interfere with attainment of the therapeutic goal.
For example, IL-6 administration has been linked to certain responses by the liver which otherwise typically characterize the mammalian "acute phase response" to a challenge such as inflammation or tissue injury. Symptoms of the acute phase response include alteration in plasma protein levels and steroid concentrations, leukocytosis, increased vascular permeability, fever, patient malaise, discomfort, fatigue, weight loss and pallor (Andus et al., FEBS Lett. 221:18 (1987)).
In particular, IL-6 has been found to act on the hepatocytes to regulate production therein of certain plasma proteins typically associated with the acute phase response, which are referred to as "acute phase proteins," see Gauldie et al., PNAS U.S.A. 84:7251 (1987); Geiger et al., Eur. J. Immunol. 18: 717 (1988)).
Such acute phase proteins include both "up-regulated" proteins, plasma levels of which are increased in response to IL-6 administration, and "down-regulated" proteins, plasma levels of which are depressed by IL-6 (see Pepys, "Acute Phase Proteins," in Encyclopedia of Immunology, Roitt, I., ed., Academic Press (1992), 16-18).
Examples of "up-regulated" acute phase proteins include a.sub.1 -antitrypsin, haptoglobulin, ceruloplasmin, alpha-1-acid glycoprotein, C-reactive protein (CRP), and alpha-2-macroglobulin. An example of a "down-regulated" protein comprises prealbumin (see Mayer et al., Exp. Hematol. 19:688-696 (1991)).
The extent of an acute phase response accompanying in vivo administration of IL-6 can be correlated to measurable changes in the serum levels of such circulating acute phase proteins.
Studies in normal rhesus monkeys demonstrate that IL-6 administration may be accompanied by a dose-related increase in serum levels of positively regulated acute phase proteins, such as CRP, alpha-1-glycoprotein, gamma-globulin, .alpha.-2-macroglobulin and fibrinogen, and likewise, a dose-related decrease in negatively regulated prealbumin, Mayer et al., id.; Ryffel et al., Toxicology Letters, 64/64 (1992), 311-319. See also Geiger et al., Eur. J. Immunol. 18:717 (1988); Castell et al., FEBS Lett. 232:347 (1988); Nijstein et al., Lancet ii:921 (1987). In Phase I trials of rhIL-6 in human cancer patients, acute phase proteins including CRP and fibrinogen increased during therapy. Olencki et al., Blood, Nov. 15, 1992, Vol. 80, No. 10, Supp. 1, #344, 346.
The occurrence of associated systemic changes comprising an acute phase response in patients can result in patient discomfort, and even become pathologic, to the point where the patient's tolerability of a drug becomes in question. A means of reducing an acute phase response can significantly improve the overall practical utility of therapeutic substances indicated to produce such response.
Granulocyte colony stimulating factor (G-CSF) has been shown to exert a regulatory effect on granulocyte-committed progenitor cells to increase circulating granulocyte levels. In particular, G-CSF can promote an increase in the number of circulating neutrophils, which assist in protecting the body against infection. Accordingly, G-CSF can be particularly useful in accelerating recovery from neutropenia in patients subjected to radiation or chemotherapy, or following bone marrow transplantation, see Dexter, "Granulocyte Colony Stimulating Factor (G-CSF), in Encyclopedia of Immunology, id.
The terms "G-CSF" and "G-CSF protein" as used herein shall be understood to refer to a natural or recombinantly prepared protein having the amino acid sequence of natural human G-CSF as disclosed, for example, in U.S. Pat. No. 4,999,291, which is incorporated herein by reference. Recombinant human G-CSF is hereinafter also referred to as "rhG-CSF".